PROJECT SUMMARY/ABSTRACT Erythropoietin (Epo) is the principle regulator of red cell production, as it controls the survival, proliferation, and differentiation of erythroid progenitors. Recombinant human Epo has been widely used to correct the anemia of renal failure as well as other types of anemias. However, diverse clinical effects of Epo in nonerythroid tissues have been reported and transcripts of EPO and/or its receptor (EPOR) genes are found in brain, endothelium, hematopoietic stem cells, breast, ovary, testes, and other tissues. Reported beneficial effects of Epo include its neuroprotective role in hypoxic stress observed in the ischemic brains of stroke patients, while other studies demonstrated that Epo administration can also be detrimental. Levels of Epo and EpoR, regulated by HIF-1 and/or HIF-2, are reported to be associated with decreased survival of cancer patients and increased cancer aggressiveness in several clinical studies, and some clinical studies of Epo therapy in cancer patient suggested that Epo administration can be detrimental. Further, EPO and EPOR transcripts have been found in some normal tissues as well as in many cancers. Due to the embryonic lethality of Epo or EpoR null mice and the absence of a proper animal model, the study of Epo function in nonerythroid cells has been limited. Previously, we generated two viable knock-in mouse models with reduced Epo signaling (these mice are anemic) and with augmented Epo signaling (these mice are polycythemic). These mice are the foundation for studying Epo function in nonerythroid tissue. We have already generated erythroid-specific Cre-expressing mice. These mice will be used to restore Epo signaling only in blood, thus rescuing the transgenic mice from anemia. These mice will be used for the generation of a gain-of-function EpoR mouse in tissues of interest such as brain, by crossbreeding with brain-specific cre mice. Hypoxia-inducible factors (HIF) are the key transcription factors that are induced by hypoxia (discovered from studies of EPO gene regulation) and that are elevated in cancer. HIFs are heterodimers that consist of an - subunit (3 isotypes; HIF-1, HIF-2, HIF-3) and a common b-subunit. HIF-1 and HIF-2 have significant homology, and while they may share regulation of some of their target genes, in some instances their gene regulation is tissue- and gene-specific. The molecular basis of HIF regulation is under intense investigation. While Epo was the first identified gene with a defined oligonucleotide sequence for HIF binding that was designated as Hypoxia Responsive Element (HRE), the EpoR gene does not have a conserved HRE. We have studied the mechanism of HIF regulation of Epo transcription using chromatin immunoprecipitation (ChIP) and have found multiple HREs of the Epo gene. Our data suggest that these HREs are differentially utilized by HIF- 1 and HIF-2 in a tissue-specific manner. Based on this evidence, we propose to study HRE in selected tissues under hypoxic and normoxic conditions using a genome-wide Chip-Seq instrument purchased by our institution. IMPACT ON VETERANS HEALTHCARE: Aging veterans, as well as newly returning veterans from Persian Gulf region, suffer from cognitive, vascular, and malignant disorders. The data obtained from this project will be used to characterize augmented Epo/EpoR signaling reported in some human malignancies and may serve as potential therapeutic cancer targets, while in other situations (i.e., strokes) the therapeutic augmentation of Epo/EpoR in selected tissues may be beneficial. Our ongoing and future planned studies should clarify the effect of Epo on atherosclerosis, hypertension, cognitive function, and prevention of ischemia-induced tissue damage.